Throughout the application various publications are referenced in parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in the application in order to more fully describe the state of the art to which this invention pertains.
1. Field of the Invention
The present invention is related to the medical arts, particularly to the field of neural tissue regeneration.
2. Discussion of the Related Art
The human nervous system comprises highly diverse cell types that make specific interconnections with one another. The nervous system includes the peripheral nerves and the central nervous system. The central nervous system includes the brain, cranial nerves, and spinal cord. Once damaged, the central nervous system of the adult has limited potential for structural self-repair. The general inability of the adult to generate new neurons (excitable cells specialized for the transmission of electrical signals from one part of the body to another) typically prevents the regeneration of neural tissues. This limitation has hindered the development of therapies for neurological injury, for example from stroke or physical trauma, or for degenerative diseases, such as Huntington disease, Alzheimer disease, and Parkinsonism. The moderate success of fetal tissue transplantation therapy for Parkinsonism suggest that cell replacement therapy can be a valuable treatment for neurological injury and degeneration.
Thus, there is a long felt need in the biomedical field for a method of generating neurons for use in the treatment of various neurological traumas, diseases, disorders, or maladies via the direct transfer of neuronal cells in a cell replacement therapy approach.
A gene therapy approach, on the other hand, is required to treat other types of nervous system disorders. Because the brain is protected by a blood-brain barrier that effectively blocks the flow of large molecules into the brain, peripheral injection of growth factor drugs, or other potentially therapeutic gene products, is ineffective. Thus, a major challenge facing the biotechnology industry is to find an efficient mechanism for delivering gene therapy products, directly to the brain, so as to treat neurological disorders on the molecular level. In this regard, a renewable source of human neural cells could serve as a vehicle to deliver gene therapy products to the brain and the rest of the central nervous system.
Until recently, the only source of donor material for these promising therapies was fetal tissue. However, the use of fetal tissue presents significant ethical and technical problems, including the limited availability of fetal tissue, the possible immuno-rejection of donor material by the recipient, and the risk of disease transmission by donor material.
Several attempts have been made to address the shortage of donor material by culturing neural progenitor cells, or neural stem cells. For example, Boss et. al taught a method for isolation and proliferation of neural progenitor cells directed to growth, storage, production and implantation of the proliferated donor cells. (Boss et. al, Proliferated Neuron Progenitor Cell Product and Process, U.S. Pat. No. 5,411,883). Anderson et. al taught a method for isolation and clonal propagation of donor mammalian neural crest stem cells capable of self renewal and differentiation into neural or glial cells. (Anderson et. al, Mammalian Neural Crest Stem Cells, U.S. Pat. No. 5,589,376). Johe taught a method for isolation, propagation and directed differentiation of stem cells from the central nervous system of embryonic and adult mammalian donors. (Johe, Isolation Propagation and Directed Differentiation of Stem Cells from Embryonic and Adult Central Nervous System of Mammals, U.S. Pat. No. 5,753,506).
Neural progenitor cells normally develop from embryonic ectodermal tissue. Bone Morphogenetic Protein (BMP) is a family of repressors that prevents ectoderm from developing into its default state of neural tissue and induces the development instead of epidermal tissue. (Y. Tanabe & T. M. Jessell, Diversity and Pattern in the Developing Spinal Cord, Science 274:1115 [1996]; Y. Sasai, Identifying the missing links: genes that connect neuronal induction and primary neurogenesis in vertebrate embryos, Neuron 21:455–58 [1998]; Y. Furuta et al, Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development, Development 124(11):2203–2212 [1997]). BMP 2 and BMP 4 induce epidermal differentiation. (E. Pera et al., Ectodermal Patterning in the Avian Embryo: Epidermis Versus Neural Plate, Development 126:63 [1999]).
BMP's can also induce cartilage formation. Hattersley et al. showed that adding BMP 13 to a cell line derived from mouse limb buds leads to the formation of chondroblast-like cells and taught a method for using BMP 13 to induce articular cartilage formation at the site of congenital or trauma induced damage and for using BMP 9 to maintain cartilage. (Hattersley et al., Cartilage Induction by Bone Morphogenetic Proteins, U.S. Pat. No. 5,902,785).
BMP signal transduction appears to be mediated by msx1, which is an immediate early response gene involved in epidermal induction and inhibition of neuronal differentiation. When Suzuki et al. injected BMP RNA into Xenopus embryos, they detected msx1 RNA production; when they injected msx1 RNA, the embryos lost neuronal structures such as eyes. (Suzuki et al., Xenopus msx1 Mediates Epidermal Induction and Neural Inhibition by BMP4, Development 124:3037 [1997]). When msx1 was added directly to dissociated ectodermal cells, epidermal development was up-regulated and neural development was down-regulated. Similarly in humans, BMP growth factors induce expression of the homeodomain transcription factor MSX1 in ectodermal cells. Once MSX1 is expressed, induction of the neuronal determination genes is simultaneously suppressed and neuronal differentiation is inhibited.
BMP seems to down-regulate neural development through at least two mechanisms: proteolysis of MASH1 protein and inhibition of Zic3 production. Exposure of neural progenitor cells to BMP triggered a rapid loss of MASH1 protein, a transcription factor that is homologous to the Drosophila Achaete-Scute Complex (ASH1) and required for the production of olfactory receptor neurons. (Shou et al., BMPs Inhibit Neurogenesis by a Mechanism Involving Degradation of a Transcription Factor, Nat. Neurosci. 2: 339 [1999]). Micro-injection of the dominant negative form of the BMP receptor, an inhibitor of BMP, into Xenopus embryos induced production of Zic3, a protein that augments neural development. (Nakata et al., Xenopus Zic3, a Primary Regulator Both in Neural and Neural Crest Development, Proc. Natl. Acad. Sci. 94: 11980 [1997]).
Antagonists of BMP signal transduction activity include fetuin glycoprotein, also known as α2-HS glycoprotein in humans, and the DAN family of BMP antagonists, such as noggin, chordin, follistatin, and gremlin. (R. Merino et al., The BMP antagonist Gremlin regulates outgrowth, chondrogenesis and programmed cell death in the developing limb, Development 126(23):5515–22 [1999]; D. Sela-Donnenfeld and C. Kalcheim, Regulation of the onset of neural crest migration by coordinated activity of BMP4 and noggin in the dorsal neural tube, Development 126(21):4749–62 [1999]). For example, Demetriou et al. showed that fetuin blocks osteogenesis, a function promoted by BMP, in a culture of rat bone marrow cells and that a fetuin derived peptide binds BMP 2. (M. Demetriou et al., Fetuin/Alpha2-HS Glycoprotein is a Transforming Growth Factor-Beta Type II Receptor Mimic and Cytokine Antagonist, J. Biol. Chem. 271:12755–61 [1996]). During embryonic and early postnatal development, Fetuin was shown to be present in a sub-population of cells in the retinal ganglion cell layer, the neuroblastic layer, and portions of the developing cerebellum. (Kitchener et al., Fetuin in Neurons of the Retina and Cerebellum During Fetal and Postnatal Development of the Rat, Int. J. Dev. Neurosci. 17: 21 [1999]).
Fetuin has been used as an additive in serum free media. Ham et al. taught the use of fetuin as an additive in serum free media for the growth of normal human muscle satellite cells directed at transplantation to the muscles of patients afflicted with muscle degenerative diseases. (Ham et al., Media for Normal Human Muscle Satellite Cells, U.S. Pat. No. 5,143,842; Ham et al., Media for Normal Human Muscle Satellite Cells, U.S. Pat. No. 5,324,656). Baker taught the use of fetuin as an additive in a defined serum free media that is capable of growing a wide range of cell suspensions and monolayers. (Baker, Serum-Free Cell Culture Medium and Process for Making Same, U.S. Pat. No. 4,560,655).
Other factors beside BMP appear to be involved in regulating neural differentiation. Ishibashi et al. demonstrated that persistent expression of Hairy and Enhancer of Split Homolog-1 (HES1) severely perturbs neuronal and glial differentiation. They infected the lateral ventricles of the brains of embryonic mice with a retrovirus that produced HES1. This led to failed migration and differentiation in the developing cells that were infected. (Ishibashi et al., Persistent Expression of Helix-Loop-Helix Factor HES-1 Prevents Mammalian Neural Differentiation in the Central Nervous System, The EMBO Journal 13: 1799 [1994]). Ishibashi et al. also disrupted the HES1 gene in mice and observed earlier than usual neurogenesis. They concluded that HES1 controls the timing of neurogenesis. (Ishibashi et al., Targeted Disruption of Mammalian Hairy and Enhancer of Split Homolog-1 (HES-1) Leads to Up-Regulation of Neural Helix-Loop-Helix Factors, Premature Neurogenesis, and Severe Neural Tube Defects, Genes &Development 9: 3136 [995]). In addition retinoids, such as retinoic acid, may play a role in inducing the differentiation of some neural cell populations. (e.g., Y. Renoncourt et al., Neurons derived in vitro from ES cells express homeoproteins characteristic of motoneurons and interneurons, Mechanisms of Development 79:185–97 [1998]).
Thus, the differentiation of neuronal tissue involves the interaction of numerous positive and negative regulatory molecules, In response to developmental signals within each cell and its surrounding microenvironment, every neuronal population expresses a specific set of neural markers, neurotransmitters, and receptors. As neural progenitor cells differentiate into other neuronal cell types in response to physiological signals in the microenvironment, the set that is expressed will be different. (E.g., see D. L. Stemple and N. K. Mahanthappa, Neural stem cells are blasting off, Neuron 18:1–4 [1997]; Y. Renoncourt et al., Neurons derived in vitro from ES cells express homeoproteins characteristic of motoneurons and interneurons, Mechanisms of Development 79:185–97 [1998]; A. J. Kalyani et al., Spinal cord neuronal precursors generate multiple neuronal phenotypes in culture, J. Neurosci. 18(19):7856–68 [1998]). Each neuronal cell type is characterized by several criteria including morphology (e.g., long processes or neurites), expression of a set of neural-specific markers (e.g., neurofilament M, neural-specific tubulin, neural-specific enolase, microtubule associated protein 2, and others), synthesis of neurotransmitters (e.g., dopamine or expression of tyrosine hydroxylase, the key enzyme in dopamine synthesis), and membrane excitability.
One of the central principles of modern neurobiology is that after differentiation each of the major projection neurons, if not all neuronal cell types, requires for its survival specific cytokines, i.e., neurotrophic or nerve growth factors, to reach their target neuronal cells. Neuropathies in many diseases may be caused by, or involve lack of, such nerve growth factors. These nerve growth factors represent the next generation of preventative and therapeutic drugs for nervous system disorders. Most of the growth factors known so far in the nervous system were discovered by their effects on peripheral nerves and these most likely represent a very minor fraction of existing growth factors in the brain. Search for growth factors from the brain has been difficult mainly because particular neuronal cell types are difficult to isolate from the brain and maintain in defined culture conditions.
Due to this limitation, drug discovery by traditional pharmacology directed to the central nervous system has been performed using whole brain homogenate and animals. These studies mostly produced analogs of neurotransmitters with broad actions and side effects. But as more and more neurotransmitter receptors and signal transducing proteins have been identified from the brain, it is becoming clear that the dogma of one neurotransmitter activating one receptor is an over-simplification. Most receptor complexes in neurons are composed of protein subunits encoded by several genes and each gene synthesizes many different variations of the protein. These variations result in a wide range of possible receptor combinations, and not a single receptor that can interact with a neurotransmitter. Consequently, a range of signal output may be produced by a single neurotransmitter action. The specific signal effected by a neurotransmitter on a neuron, then, depends on which receptor complex is produced by the cell. Thus, cellular diversity must parallel the molecular diversity and constitute a major structural element underlying the complexity of brain function, and a source of diverse neuronal cell types that can be cultured for drug screening purposes is needed.
Therefore, there remains a need in the field of neurological research and applied neurobiology for a renewable non-fetal source of neural progenitor cells and cells having characteristics specifically associated with neuronal or glial cell types, for use in research, cell therapy, or gene therapy. Importantly, the use of such cells could eliminate a need for fetal human tissue in therapeutic approaches aimed at restoring neurological function by intracerebral transplantation of nervous system cells. These and other benefits the present invention provides as described herein.